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Raw milk cheeses harbor complex microbial communities. Some of these microorganisms are technologically essential, but undesirable microorganisms can also be present. While most of the microbial dynamics and cross-talking studies involving interaction between food-derived bacteria have been carried out on agar plates in laboratory-controlled conditions, the present study evaluated the modulation of the resident microbiota and the changes of metabolite production directly in ripening raw milk cheese inoculated with Listeria innocua strains. Using a proxy of the pathogenic Listeria monocytogenes, we aimed to establish the key microbiota players and chemical signals that characterize Latteria raw milk cheese over 60 days of ripening time. The microbiota of both the control and Listeria-inoculated cheeses was analyzed using 16S rRNA targeted amplicon sequencing, while direct analysis in real time mass spectrometry (DART-HRMS) was applied to investigate the differences in the metabolic profiles of the cheeses. The diversity analysis showed the same microbial diversity trend in both the control cheese and the inoculated cheese, while the taxonomic analysis highlighted the most representative genera of bacteria in both the control and inoculated cheese: Lactobacillus and Streptococcus. On the other hand, the metabolic fingerprints revealed that the complex interactions between resident microbiota and L. innocua were governed by continuously changing chemical signals. Changes in the amounts of small organic acids, hydroxyl fatty acids, and antimicrobial compounds, including pyroglutamic acid, hydroxy-isocaproic acid, malic acid, phenyllactic acid, and lactic acid, were observed over time in the L. innocua-inoculated cheese. In cheese that was inoculated with L. innocua, Streptococcus was significantly correlated with the volatile compounds carboxylbenzaldheyde and cyclohexanecarboxylic acid, while Lactobacillus was positively correlated with some volatile and flavor compounds (cyclohexanecarboxylic acid, pyroxidal acid, aminobenzoic acid, and vanillic acid). Therefore, we determined the metabolic markers that characterize a raw milk cheese inoculated with L. innocua, the changes in these markers with the ripening time, and the positive correlation of flavor and volatile compounds with the resident microbiota. This multi-omics approach could suggest innovative food safety strategies based on the enhanced management of undesirable microorganisms by means of strain selection in raw matrices and the addition of specific antimicrobial metabolites to prevent the growth of undesirable microorganisms. [ABSTRACT FROM AUTHOR]

Aims: The efficacy of ambient mass spectrometry to identify and serotype Legionella pneumophila was assessed. To this aim, isolated waterborne colonies were submitted to a rapid extraction method and analysed by direct analysis in real‐time mass spectrometry (DART‐HRMS). Methods and Results: The DART‐HRMS profiles, coupled with partial least squares discriminant analysis (PLS‐DA), were first evaluated for their ability to differentiate Legionella spp. from other bacteria. The resultant classification model achieved an accuracy of 98.1% on validation. Capitalising on these encouraging results, DART‐HRMS profiling was explored as an alternative approach for the identification of L. pneumophila sg. 1, L. pneumophila sg. 2–15 and L. non‐pneumophila; therefore, a different PLS‐DA classifier was built. When tested on a validation set, this second classifier reached an overall accuracy of 95.93%. It identified the harmful L. pneumophila sg. 1 with an impressive specificity (100%) and slightly lower sensitivity (91.7%), and similar performances were reached in the classification of L. pneumophila sg. 2–15 and L. non‐pneumophila. Conclusions: The results of this study show the DART‐HMRS method has good accuracy, and it is an effective method for Legionella serogroup profiling. Significance and Impact of the Study: These preliminary findings could open a new avenue for the rapid identification and quick epidemiologic tracing of L. pneumophila, with a consequent improvement to risk assessment. [ABSTRACT FROM AUTHOR]